Investigations within this project concern the cell biology of rare human genetic disorders and normal and abnormal intracellular processes. The research goal is to gain insight into changes in molecular function that underlie various genetic metabolic disorders and work towards treatments for these illnesses. The research focuses on four groups of rare disorders: 1. Disorders of sialic acid metabolism. The key enzyme in the sialic acid biosynthesis pathway is UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Dominant mutations in the allosteric site of GNE cause sialuria, characterized by overproduction of sialic acid. Recessive mutations in GNE cause the neuromuscular disorder hereditary inclusion body myopathy (HIBM). In 2007, we characterized a knock-in HIBM mouse model and demonstrated that N-acetylmannosamine (ManNAc) rescues the phenotype of the homozygous mutant mice and is a promising treatment option for human patients (Galeano et al. J Clin Inv (2007) 117:1585-1594). Negotiations regarding an extensive toxicology study for the use of ManNAc are ongoing, and our ManNAc patent is in the process of being transferred to New Zealand Pharmaceuticals, a ManNAc manufacturor. We are currently testing other treatment options on our murine HIBM model, including feeding studies with sialic acid pathway intermediates and GNE gene therapy. The feeding studies have been completed with sialic acid, mannose, mannosamine, as well as butinylated ManNAc and a manuscript is in preparation. A pilot gene therapy study with a healthy GNE gene embedded in liposomes (GNE-Lipoplex) was performed with promising results, this method is now further examined to rescue the kidney and muscle phenotypes of our HIBM mice. Our murine HIBM model showed an unexpected kidney phenotype (of podocytopathy and glomerular membrane splitting) which was rescued by ManNAc feeding. We developed a lectin panel that characterized the glycosylation status of our HIBM mouse model. We are currently testing this panel on a variety of unexplained human renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane. These human disorders may benefit from ManNAc as a therapeutic agent. Furthermore, our group performs GNE mutation analyses for collaborating research groups (Refs 9,10), and we are investigating tissue distribution (by real-time PCR and Western blotting) and folding (by molecular modeling) of newly discovered GNE isoforms in human and mice. We wrote an extensive review on all aspects of HIBM (Ref 7). 2. Disorders of 3-methylglutaconic aciduria (3MGA) presenting with or without optic atrophy. In 2001, our group isolated OPA3, a gene of unknown function responsible for Costeff syndrome, which is characterized by 3MGA and optic atrophy. We tested DNA from patients with/without 3MGA and/or isolated optic atrophy for mutations in OPA3 (Ref 15). We investigated OPA3 function, and identified a novel OPA3 isoform with a rare dual mitochondrial and peroxisomal localization. We also created zebrafish models for Costeff syndrome using antisense morpholino technology (manuscripts submitted). 3. Disorders of intracellular vesicle sorting and formation. These disorders include Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, and other genetically unclassified disorders. Common clinical features are albinism due to defects in melanosomes and bleeding due to platelet defects. Our group investigates known and unknown HPS-causing genes, with the goal of better understanding the biology of the disease. Our group also catalogues the clinical and genetic characteristics of the seven distinct subgroups of HPS. And we perform candidate gene screening on unclassified patients (Refs 5,8,12,14). To study the effects of HPS mutations, we perform cell biological studies on patients material (employing immuno-fluorescence, immmuno-EM, and live cell imaging) to examine defective intracellular trafficking and sorting of proteins and organelles in HPS cells. Such cells fail to transport certain lysosomal proteins to their correct destinations, and HPS gene products are involved in recognizing the specific vesicles that give rise to lysosome-like organelles (Refs 2,3,11). 4. Genetic interstitial deletion syndromes. Routine mutation screening commonly involves PCR-based approaches followed by direct sequencing. Occurrence of larger genomic deletions may be missed by these approaches if the deletion breakpoints extend beyond the position of the PCR primers. In recessive disorders, this can lead to mistaking homozygosity for hemizygosity. Our group applied quantitative real-time PCR to detect hemizygosity and deletion breakpoints in a variety of rare disorders. - Hermansky-Pudlak syndrome: We identified patients with a large genomic deletion on the HPS1 locus (Griffin et al. Clin Genet (2005) 68, 23-30) and the HPS6 locus manuscript (under revision J Med Genet). - Holoprosencephaly: We tested patients by quantitative real-time PCR for submicroscopic deletions in candidate gene regions (Bendavid et al. J Med Genet (2006) 43, 496-500), supplementing multicolor FISH results. - Smith-Magenis syndrome (SMS): This disorder is mainly (greater than 95%) caused by an interstitial deletion of 17p11.2. Our group is currently performing quantitative real-time PCR to identify hemizygosity in key genes on 17p11.2 in 98 patients with SMS. Our results, in combination with FISH analysis and comparative genome hybridization (CGH)- arrays performed by collaborating groups, will shed light on the variable phenotype of SMS patients and genotype-phenotype correlations (Ref 4, other manuscripts in preparation). In the future, these quantitative real-time PCR methods can be applied to other deletion syndromes (e.g., Jacobsen syndrome). - FISH and qPCR analysis on 20 SMS patients identified no deletion in 17p11.2. Mutation analysis for single gene defects are ongoing (including RAI1 and RASD1) in our lab. We also performed CGH-arrays on these patients DNA to identify novel (micro) deletions or duplications. Preliminary results identified 4 novel chromosomal rearrangements (manuscript in preparation).